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A Comparative Transcriptomic Analysis of Human Placental Trophoblasts in Response to Pathogenic and Probiotic Enterococcus Faecalis Interaction

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A Comparative Transcriptomic Analysis of Human Placental Trophoblasts in Response to Pathogenic and Probiotic Enterococcus Faecalis Interaction Hindawi Canadian Journal of Infectious Diseases and Medical Microbiology Volume 2021, Article ID 6655414, 9 pages https://doi.org/10.1155/2021/6655414 Research Article A Comparative Transcriptomic Analysis of Human Placental Trophoblasts in Response to Pathogenic and Probiotic Enterococcus faecalis Interaction Qianglai Tan,1,2 Zhen Zeng,1 Feng Xu,2 and Hua Wei 2 1Xiamen Medical College, Xiamen 361023, Fujian, China 2State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, Jiangxi, China Correspondence should be addressed to Hua Wei; [email protected] Received 5 October 2020; Revised 17 December 2020; Accepted 12 January 2021; Published 28 January 2021 Academic Editor: Maria De Francesco Copyright © 2021 Qianglai Tan et al. ,is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. With the ability to cross placental barriers in their hosts, strains of Gram-positive Enterococcus faecalis can exhibit either beneficial or harmful properties. However, the mechanisms underlying these effects have yet to be determined. A comparative tran- scriptomic analysis of human placental trophoblasts in response to pathogenic or probiotic E. faecalis was performed in order to investigate the molecular basis of different traits. Results indicated that both E. faecalis Symbioflor 1 and V583 could pass through the placental barrier in vitro with similar levels of invasion ability. In total, 2353 (1369 upregulated and 984 downregulated) and 2351 (1233 upregulated and 1118 downregulated) DEGs were identified in Symbioflor 1 and V583, respectively. Furthermore, 1074 (671 upregulated and 403 downregulated) and 1072 (535 upregulated and 537 downregulated) DEGs were only identified in Symbioflor 1 and V583 treatment groups, respectively. KEGG analysis showed that 6 and 9 signaling pathways were associated with interactions between Symbioflor 1 and V583. GO analysis revealed that these DEGs were mainly related to cellular and metabolic processes and biological regulation. However, 28 and 44 DEGs were classified into terms associated with placental and embryonic development in Symbioflor 1 and V583 treatment groups, respectively. Notably, 9 and 25 unique DEGs were identified only in Symbioflor 1 and V583 treatment groups, respectively. A large proportion of transcriptional responses differed when compared between pathogenic and probiotic E. faecalis interaction, and several unique DEGs and signal pathways were identified in the two different groups. ,ese data enhance our understanding of how different traits can be affected by pathogenic and probiotic E. faecalis and the mechanisms underlying these effects. 1. Introduction et al. confirmed that various enterococci could transmit to fetus and cause stillbirth [6]. We have previously demon- Enterococcus faecalis is a Gram-positive bacterium that is strated that E. faecalis OG1RF can pass through the placental commonly found in a variety of different matrices including barrier of pregnant mice, translocate into the fetus, and then the alimentary tract and foods. ,is bacterium has received affect both fetal and placental growth and development [7]. substantial attention due to the fact that it can exert both We demonstrated that E. faecalis OG1RF induces placental beneficial and pathogenic effects [1]. Certain E. faecalis and embryonic development retardation, stress and stimulus strains are regarded as commensal bacteria or even pro- response activation, apoptosis, immune response disorder, biotics for creating a healthy gut environment; however, and cell adhesion disintegration of placental trophoblasts other strains are considered to be dangerous as they can lead through various signaling pathways using real-time PCR and to a variety of nosocomial infections and diseases [2, 3]. DNA microarray [8]. E. faecalis has also been identified in Previous researches have shown that E. faecalis is able to pass meconium obtained from healthy neonates. ,e presence of through biological barriers and can subsequently mediate a this type of bacteria in meconium could initiate gut colo- variety of effects in the host [4, 5]. A review by Goldenberg nization as an adaptation to the fetal gut to prepare the fetus 2 Canadian Journal of Infectious Diseases and Medical Microbiology ° for life outside the mother [9]. Albesharat et al. found that 37 C under a 5% CO2 atmosphere, until approximately 80%– E. faecalis was present in the feces of mothers and their 90% cell confluence. babies and hypothesized that the initial bacterial coloniza- tion of the infant occurs via vertical transmission from mothers to infants [10]. ,e close relationship between two 2.2. Invasion Assays. Invasion assays were performed in typical E. faecalis strains (Symbioflor 1 [11] and V583 [12]) accordance with our previous study [8]. BeWo cells were has been studied in significant detail with regards to human transferred into 24-well plates (Corning, NY) and cultured pregnancy. E. faecalis Symbioflor 1 (SymbioPharm, Her- for 24–48 h until a confluent monolayer was obtained. A born, Germany) is recognized as a probiotic strain that can 5 μL aliquot of resuspended bacteria was then added to each alleviate the symptoms of irritable bowel syndrome by well containing 495 μL of cell culture medium and incubated improving the microbial balance in the intestine. E. faecalis for 60 minutes. V583 is a vancomycin-resistant prototype clinical isolate ,e total number of invading bacteria was determined by that causes opportunistic nosocomial infection worldwide. twice dip-washing with sterile PBS to remove free bacteria Meanwhile, a human placental choriocarcinoma BeWo cell and lysing the BeWo cells with 500 μL of 0.5% Triton X-100/ lines, a widely recognized in vitro placental model [13], was PBS to release the internalized bacteria. Serial dilutions were adopted. ,e BeWo cell line is regularly used to study a range spread onto Trypticase soy agar (TSA) plates and incubated of placental functions, including transplacental transport at 37°C overnight. ,e numbers of internalized bacteria were and infection caused by viruses and bacteria. DEGs of BeWo then determined by adding 100 μg/mL of gentamicin and cells associated with E. faecalis invasion were identified by 50 μg/mL of penicillin and incubating for 60 min to kill any using DNA microarray, and transcriptomic profiling was viable extracellular bacteria that were still present. ,e cells compared by using Gene Ontology (GO) and Kyoto En- were then washed twice, lysed, and the bacterial count cyclopedia of Genes and Genomes (KEGG) pathway data- determined as described above. P values were calculated base [14]. using Studentʼs t-test. In this study, we first constructed an interaction model between two different E. faecalis strains and BeWo cells. ,en, we compared the invasion abilities (adhesion ability 2.3. DNA Microarray and Data Analysis. Incubation ex- and internalization ability) between the two E. faecalis periments were performed as described in our previous strains. Subsequently, we performed comparative tran- study [8]. In brief, BeWo cells were grown in 24-well plates scriptomic profiling by using a DNA microarray (Illumina and cultured for 24–48 h until 80%–90% confluency. Human HT-12 v4 Expression Bead Chip), GO analysis, and E. faecalis V583 and Symbioflor 1 were then harvested and KEGG pathway analysis. ,e main objective of this study resuspended in DMEM/F-12 cell culture medium to was to gain insights into the differences in transcriptional 109 CFU/mL. A 5 μL aliquot of resuspended E. faecalis regulation between strains of E. faecalis that can exhibit V583 and Symbioflor 1 was then added to each well dualistic behavior towards host health. ,e study was containing 495 μL of cell culture medium and incubated for designed to identify the different traits that might be affected 4 h. ,e same amount of cell culture medium, but without by putative pathogenic or probiotic E. faecalis. ,e identi- any E. faecalis strains, was used as a negative control. Total fication of such traits is crucial as this information may RNA was extracted using TRNzol Total RNA Reagent ultimately contribute to the future development of strategies (TIANGEN, Beijing, China). DNA microarrays were for the prevention and treatment of invasion and infection performed by Beijing EMTD Technology Development caused by different E. faecalis strains. Co., Ltd. using an Illumina Human HT-12 v4 Expression BeadChip system (Illumina, Inc., San Diego, CA). In brief, 2. Materials and Methods RNA was adjusted to a concentration of 200 ng/μL, fol- lowed by first and then second strand cDNA synthesis. 2.1. Bacterial Strains and Cell Lines. ,e bacterial strains Double-stranded cDNA was then purified with a filter used in this study are listed in Table 1. In brief, strains of cartridge, and cRNA was synthesized by T7 RNA poly- E. faecalis strains, clinical pathogen V583 (ATCC 700802), merase transcription in vitro. Following purification and and probiotic Symbioflor 1 (DSM 16431) were cultured in quantification, the cRNA was hybridized with the Bead- Trypticase soy broth (TSB) at 37°C for 24 h with shaking at Chip, washed, scanned, and analyzed. Illumina expression 180 rpm. ,e bacteria were then harvested by centrifugation data were deposited in the NCBI Gene Expression Om- at 6000 rpm for 10 min, washed twice with sterile PBS nibus (GEO) database under the accession number (0.01M, pH 7.4), and reconstituted in cell culture medium GSE75626. DEGs were analyzed using GO and KEGG DMEM/F-12 (Solarbio, Beijing, China) to yield a concen- pathway databases. tration of 109 CFU/mL before use. ,e human choriocarcinoma cell line BeWo was pur- chased from Action-award Biotech Co., Ltd. (Guangzhou, 2.4. Statistical Analysis. Unless specified, all experiments China) and cultured as described previously [8]. In brief, were performed in triplicate. All data were analyzed using BeWo cells were cultured in DMEM/F-12 medium sup- statistics programs contained in SigmaPlot 11.0 (Systat plemented with 10% (v/v) FBS (Gibco, Grand Island, NY) at Software, San Jose, CA).
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